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Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids
Published on: September 21, 2017
RModBlock antisense oligonucleotides as a universal tool for precise and efficient inhibition of RNA modifications
Mengdan Ma1,2, Jing Yao1,2, Wanying Chen3
1MOE Key Laboratory of Gene Function and Regulation, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China.
Abstract:
The ability to selectively edit specific RNA modifications is needed to understand their roles in cellular function and disease. However, existing approaches, particularly catalytically inactive Cas (dCas)-based systems, have limited applicability owing to their reliance on eraser proteins, and their performance can vary depending on the modification type and target context. Here we introduce the RNA Modification-Blocking (RModBlock) strategy, which uses chemically modified antisense oligonucleotides (ASOs) with locked nucleic acid to precisely inhibit modifications at targeted sites. Most RNA modification writers, including those for m5C and pseudouridine, require specific structural contexts. Using representative modifications with and without eraser proteins, m5C and pseudouridine, respectively, we demonstrated that RModBlock ASOs blocked the formation of their structural context, inhibiting modified bases by up to 97% in human cells. RModBlock ASOs also inhibited m6A, despite its writer protein not requiring a strict structural context, suggesting broad applicability. Moreover, this strategy achieves comparable or superior performance when dCas13-eraser-based systems are applicable. Finally, by inhibiting cancer-relevant modifications and through in vivo delivery to the mouse liver, we highlight its therapeutic potential. This showcases the RModBlock strategy as a precise, efficient and versatile approach for manipulating RNA modifications, with broad applicability in basic and translational research.
Insights
Researchers developed a new RNA modification-blocking (RModBlock) strategy using chemically modified antisense oligonucleotides. This precise method effectively inhibits RNA modifications, showing significant potential for basic research and therapeutic applications.
Area of Science:
- Molecular Biology
- Epigenetics
- RNA Biology
Background:
- Understanding RNA modifications is crucial for cellular function and disease research.
- Current methods, like dCas-based systems, have limitations in applicability and performance depending on modification type and context.
- Targeted inhibition of RNA modification writers is needed for precise manipulation.
Purpose of the Study:
- To introduce and validate the RNA Modification-Blocking (RModBlock) strategy for precise inhibition of RNA modifications.
- To assess the efficacy of RModBlock ASOs across different modification types (m5C, pseudouridine, m6A).
- To evaluate the therapeutic potential of RModBlock strategy in cellular and in vivo models.
Main Methods:
- Development of chemically modified antisense oligonucleotides (ASOs) incorporating locked nucleic acid for targeted RNA modification inhibition.
- Application of RModBlock ASOs to block structural contexts required by RNA modification writers.
- Testing RModBlock efficacy on m5C, pseudouridine, and m6A modifications in human cells.
- In vivo delivery of RModBlock ASOs to mouse liver.
Main Results:
- RModBlock ASOs effectively inhibited m5C and pseudouridine formation by up to 97% by blocking their required structural contexts.
- The strategy also successfully inhibited m6A modification, demonstrating broad applicability.
- RModBlock performance was comparable or superior to existing dCas13-eraser systems.
- Inhibition of cancer-relevant modifications and successful in vivo delivery highlighted therapeutic potential.
Conclusions:
- The RModBlock strategy offers a precise, efficient, and versatile approach for manipulating RNA modifications.
- This method has broad applicability in both basic science and translational research.
- RModBlock holds promise as a therapeutic tool for diseases associated with aberrant RNA modifications.
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